Currently, the mouse is the model system of choice for cell transplantation assays (Greiner, et al., 1998). Cells are injected into adolescent or adult mice followed by injection of compounds and examination for viability and tumor size (Yang, et al., 1997; Katsanis et al., 1998). To prevent cell rejection, transplantation of human cells must be performed using immunosuppressed mice from either Nude or SCID mouse lines (Yang, et al., 1997;Greiner, et al., 1998;Katsanis et al., 1998). However, because these mice do not exhibit an immune response, they are less hardy than normal mice and more susceptible to toxic effects of the compounds. In addition, these animals are expensive to develop and maintain. Furthermore, because mice develop en utero, it is not possible to assay mouse embryos, greatly complicating assessment of the effect of compounds on developmental processes. A hollow fiber model, in which tiny tubes filled with tumor cells are implanted into mice in a variety of sites is also used for drug screening. By monitoring the tumor cell killing effects of drugs on the implants, researchers can test which drugs actually make it to the tumor sites when the drugs are administered in different ways: intravenously versus orally, for example.
Limitations of animal models have spurred the NCI and others to also test drug candidates in cultures of human cells and the Institute now relies on a panel of 60 human tumor cell lines, including samples of all the major human malignancies. Drugs to be tested are fed to subsets of the panel, based on tumor cell type and their cell killing activity is monitored.
Clonogenic assays are also performed. In this method, cell lines or a patient""s tumor cells are grown in petri dishes or culture flasks and the cell""s responses to various anticancer treatments are monitored. However, these assays are also problematic. Sometimes they do not work because the cells simply fail to divide in culture. Furthermore, results do not predict how an anticancer drug will perform in the body.
In a continuing search for faithful models of human carcinogenesis, NCI has recently begun reclassifying the cells based on tissue type-breast cancer versus colon cancer, for example, according to the types of genetic defects the cells carry. To enable drugs that counteract specific defects to be prescribed most effectively, researchers are also developing technologies for analyzing the gene defects in each patients"" tumors in order to determine if drugs that correct specific defects can be identified, since they could then be matched to each individual tumor cell makeup.
To create better models of cancer development in humans, investigators are now drawing on the growing knowledge of human cancer related gene mutations. They are genetically altering mice so that they carry the same kinds of changes either abnormal activation of cancer promoting oncogenes or loss of tumor suppressor genes that lead to cancer in humans. The hope is that the mice will develop tumors that behave the same way the human tumors do. One mutant mouse strain, for example lacks a working APC gene, a tumor suppressor that lead to colon cancer when lost or inactivated. So far the results have been mixed.
The invention provides methods of cellular analysis using fish. Such methods entail introducing one or more heterologous cells into a fish, and analyzing a property of the cells or the fish. The methods are particularly suited for introduction of heterologous cells into fish embryos, particularly zebrafish embryos. Introduced cells remain viable at least until the analyzing step is performed. Some cell types undergo proliferation in the recipient fish. In some methods, the fish is contacted with an agent, and the analyzing determines whether the property is responsive to administration of the agent. Properties of heterologous cells or fish that can be analyzed include differentiation markers, r, survival of the fish, proliferation of the heterologous cells, movement of the heterologous cells relative to an initial site of introduction, death of heterologous cells or cells of the fish, or proliferation of heterologous cells. In some methods, the heterologous cells are cancer cells. In some methods, the heterologous cells are stem cells. In some methods, the heterologous cells are differentiated cells. In some methods, the heterologous cells are human cells. In some methods, the heterologous cells are bacterial or fungal cells. In some methods, the cells are virally infected cells. Some methods further comprising recovering heterologous cells from recipient fish.
The invention further provides methods of screening an agent for activity against cancerous cells. Such methods entail introducing one or more cancerous cells into a population of fish, administering the agent to the population of fish, and monitoring an effect of the agent on development of the cancerous cells in the population of fish. In some methods, the monitoring step comprises determining an EC50 for the effect of the agent on development of the cancerous cells in the fish. In some methods, the monitoring step comprises detecting an LD50 of the agent on the population of fish. Optionally, the method is repeated for a plurality of agents, and an agent with a low EC50|LD50 ratio is formulated with a carrier as a pharmaceutical composition.
The invention further provides methods of propagating cells. Such methods entail introducing one or more heterologous cells into a fish, culturing the fish under conditions in which the cells proliferate; and recovering the proliferated cells. In some methods, the cells differentiate in the course of proliferation, and the cells are recovered as a differentiated tissue. In some methods, recovered cells are transplanted into a patient, optionally the same patient from whom the heterologous cells were obtained.
The invention further provides methods of diagnosing a sample for a cancerous cell or pathogen. Such methods entail obtaining a sample from a patient containing a population of cells; introducing the population of cells into a fish; and detecting a property of the population of cells to indicate whether the population comprises a cancerous cell or pathogen.